bioRxiv preprint doi: https://doi.org/10.1101/2020.03.24.002816; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Transcriptional network analysis of transcriptomic diversity in resident tissue macrophages and dendritic cells in the mouse mononuclear phagocyte system. Kim M. Summers*, Stephen J. Bush# and David A. Hume* *Mater Research Institute-University of Queensland, Translational Research Institute, WoolloonGabba, Qld 4012, Australia and #Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK. The three authors contributed eQually to this work. Address for correspondence Professor David Hume Mater Research Institute-University of Queensland Translational Research Institute 37 Kent Street WoolloonGabba, Qld 4102 Australia [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.24.002816; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract The mononuclear phaGocyte system (MPS) is a family of cells includinG proGenitors, circulatinG blood monocytes, resident tissue macrophaGes and dendritic cells (DC) present in every tissue in the body. To test the relationships between markers and transcriptomic diversity in the MPS, we collected from NCBI-GEO >500 Quality RNA-seQ datasets Generated from mouse MPS cells isolated from multiple tissues. The primary data were randomly down-sized to a depth of 10 million reads and requantified. The resultinG dataset was clustered using the network analysis tool Graphia. A sample-to-sample matrix revealed that MPS populations could be separated based upon tissue of origin. Cells identified as classical DC subsets, cDC1 and cDC2, and lacking Fcgr1 (CD64), were centrally-located within the MPS cluster and no more distinct than other MPS cell types. A Gene-to-gene correlation matrix identified large Generic co-expression clusters associated with MPS maturation and innate immune function. Smaller co-expression clusters including the transcription factors that drive them showed higher expression within defined isolated cells, includinG macrophaGes and DC from specific tissues. They include a cluster containinG Lyve1 that implies a function in endothelial cell homeostasis, a cluster of transcripts enriched in intestinal macrophaGes and a generic cDC cluster associated with Ccr7. However, transcripts encodinG many other putative MPS subset markers includinG Adgre1, Itgax, Itgam, Clec9a, Cd163, Mertk, Retnla and H2-A/E (class II MHC) clustered idiosyncratically and were not correlated with underlying functions. The data provide no support for the concept of markers of M2 polarization or the specific adaptation of DC to present antigen to T cells. Co-expression of immediate early genes (e.g. Egr1, Fos, Dusp1) and inflammatory cytokines and chemokines (Tnf, Il1b, Ccl3/4) indicated that all tissue disaGGreGation protocols activate MPS cells. Tissue-specific expression clusters indicated that all cell isolation procedures also co-purify other unrelated cell types that may interact with MPS cells in vivo. Comparative analysis of public RNA-seQ and sinGle cell RNA-seQ data from the same lunG cell populations showed that the extensive heteroGeneity implied by the Global cluster analysis may be even greater at a single cell level with few markers stronGly correlated with each other. This analysis hiGhliGhts the power of larGe datasets to identify the diversity of MPS cellular phenotypes, and the limited predictive value of surface markers to define lineages, functions or subpopulations. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.24.002816; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Introduction The mononuclear phaGocyte system (MPS) [1] is a family of cells present in every tissue in the body includinG proGenitors, circulatinG blood monocytes and resident tissue macrophaGes [2-5]. Within each tissue, resident macrophages occupy territories with a reGular distribution, commonly associated with epithelial and endothelial surfaces (reviewed in [5]). The proliferation, differentiation and survival of most resident macrophaGe populations depends upon siGnals from the macrophaGe-colony-stimulatinG factor receptor (CSF1R) initiated by one of two ligands, CSF1 or IL34 [6, 7]. Based upon detection of macrophaGe-restricted mRNA, includinG Csf1r, the relative abundance of resident macrophaGes in most organs in mice was shown to reach a maximum in the first week of postnatal life and remains stable thereafter durinG postnatal Growth [8]. LineaGe-trace studies in the C57BL/6 strain suGGest that many macrophaGe populations established in the mouse embryo are maintained in adults mainly by self- renewal, whereas others are replaced proGressively to differinG extents by blood monocytes derived from bone marrow proGenitors throuGhout life [9-11]. Most if not all tissue macrophaGe populations can be generated and maintained in the absence of blood monocytes due to the intrinsic homeostatic reGulation by circulatinG CSF1 [12]. The precise details of ontogeny, turnover and homeostasis of resident macrophaGes may not be conserved across mouse strains or species [5]. However, reGardless of their steady-state turnover, all resident macrophaGes includinG the microGlia of the brain can also be rapidly replaced by blood monocytes followinG experimental depletion ([3-5, 12]and references therein). Within individual tissues, resident macrophaGes acQuire specific adaptations and Gene expression profiles [2, 5, 13-15]. These adaptations contribute to survival as well as function and involve inducible expression of transcription factors and their downstream target genes. At least some of these transcription factors act by reGulatinG Csf1r expression. Deletion of a conserved enhancer in the mouse Csf1r gene leads to selective loss of some tissue macrophaGe populations, whereas others express Csf1r normally [16]. In the mouse embryo, where abundant macrophaGe populations are enGaGed with phagocytosis of apoptotic cells [17], the macrophaGe transcriptome does not differ Greatly between organs. Tissue-specific macrophaGe adaptation occurs mainly in the postnatal period as the organs themselves exit the proliferative phase and start to acquire adult function [8, 15]. Classical dendritic cells (cDC) are commonly defined functionally on the basis of a proposed unique ability to present antigen to naïve T cells, a concept that reQuires a clear distinction between DC and macrophaGes [18]. It remains unclear as to whether cDC should be considered part of the MPS and the extent to which they can be defined by surface markers [12]. The situation is confused by the widespread use of the term DC to describe any antigen-presenting cell (APC) includinG cells that are clearly derived from blood monocytes [19]. An attempt at consensus proposed an MPS nomenclature classification based upon ontogeny, and secondarily upon location, function and phenotype [20]. The proposal separates monocyte-derived APC from cDC subsets; cDC1, dependent on the transcription factor BATF3, and cDC2, dependent upon IRF4. Some support for this separation came from analysis of an Ms4a3 reporter transgene, which labelled cells derived from committed granulocyte-macrophaGe progenitors and distinguished monocyte-derived cells from tissue DC [10]. Secondary classification is based upon cell surface markers that are presumed to be linked in some way to ontogeny. The proposed development pathway of these DC subsets from a common myeloid progenitor, via a common DC progenitor (CDP) has been reviewed recently [21]. Even within tissues resident macrophaGes are extremely heterogeneous [22, 23]. Since the advent of monoclonal antibodies and later development of transgenic reporter Genes [24] numerous markers have been identified that segregate the MPS into subpopulations. AmonGst the recent suGGestions, LYVE1 was proposed as a marker of macrophages associated with the vasculature [25], CD64 (Fcgr1gene) and MERTK as markers that distinGuish macrophaGes from classical DC [26, 27] and CD206 (Mrc1 gene) a marker of so-called M2 macrophage polarization [28]. Several surface markers have also been identified that are encoded by genes expressed only in macrophages in specific tissues (e.g. Clec4f, Tmem119, Siglecf [22, 23]. Other markers define macrophaGes in specific locations with a tissue, for example CD169 (encoded by Siglec1) in the marginal zone of spleen and hematopoietic islands in bone marrow [29]. In the case of blood monocytes, the subpopulations are clearly a differentiation series in which short-lived LY6Chi “classical” monocytes give rise in a CSF1R-dependent manner [30] to lonG- lived LY6Clo non-classical monocytes via an intermediate state [11, 30, 31]. This is likely also the case 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.24.002816; this version posted March 25, 2020. The copyright holder for this